The most frequent cause of a failed DLW study is poorly sealed capillaries.
It is important to learn how to seal capillaries in the lab BEFORE going out in the field. Try with water first (or ink as it's easier to see) as blood is more difficult. Fill the capillary to the half way mark, and then rock it so that the water/ink moves to the centre of the tube. You should hold your thumb and finger of your left hand (if you are right handed) over the sample, so that only glass is poking out. It is a good idea to position your hand so that the other end is not pointing directly into your palm (see Fig a below. This figure is from Speakman 1997 doubly-labelled water theory and practice. Artwork by Susan Thomson) in case the sample gets heated and shoots into your palm – it is easiest to do this if you hold the capillary from beneath (think - the 'ohm' position when doing yoga). The reason for putting your finger and thumb over the sample is that you want to avoid heating the sample – if you don't burn your fingers you won't heat the sample. Heat the capillary using the hottest part of the flame (at the top of the blue cone) at a distance of ~2cm from your fingers. Hold the free end of the capillary with a pair of tweezers or your other hand.
Once the capillary glass starts to melt (b), roll the capillary ½ -1 turn between your thumb and finger (in one direction). Keep the forceps still so that the capillary rotates and the molten glass collapses (think of how water in a hose pipe stops if you get a kink in it – rolling the capillary twists the glass as it melts forming a better seal). The biggest problem people appear to have, is not timing when to separate the two pieces of the capillary. If you pull your hands apart too fast, you tend to get long glass fibres generated from both ends of the capillary tubes. These are potentially hazardous, not to mention the fact that the tube does not seal well. If you find you are getting these, take more time and leave your capillary in the flame a little longer before you draw the two pieces apart (c). If you find the end of the capillary blows up into a bubble, then you are leaving the end in the flame too long. In this circumstance the capillary seals, but then you leave it there and the air inside expands under the heat, blowing a bubble in the molten glass. These bubbles often smash later as they are very thin walled and your sample will be lost as it dries out. If the ends you generate on your capillaries are bent, the capillary will not fit in the distillation tube, so keep your hands level as you move them apart (c).
Another common problem is pulling the pieces apart at the same time as taking them out of the flame - the glass cools down too quickly and fails to separate. Finally, many people tend to pull their arms too far apart. This also leads to long glass fibres. Try to restrict pulling your hands apart to less than 10 cm. If you pull your hand holding the sample away from the flame by ~2cm as the capillary begins to melt and then leave it in the flame to separate you should get nice rounded ends, because you are positioning solid glass (where the capillary has collapsed) into the flame. This should melt without forming a bubble.
Do not pull the hand holding the tweezers away from the flame, because if the capillary has not melted enough, you may pull your sample into the flame, or at least re-position the capillary so that the flame is heating the air next to the sample which will cause a bubble to form. Once you have sealed one side, you can turn it around and seal the other side (d and e). Warning - the side that you have just sealed will be hot! This may seem obvious, but people always forget and burn themselves. Once you have turned the capillary around, you can repeat the procedure on the other end - again keeping you finger and thumb over the sample. When it works the sample should keep indefinitely.
Sealing blood into capillaries is more difficult than encapsulating water because of a reaction that the blood has with the glass when it is heated. When you draw up a blood sample into the capillary and position the sample in the centre there are two distinct sides to the capillary. The dirty side through which the sample has passed, and the clean side through which nothing has passed. The dirty side has blood smeared on the inside of the tube. Since blood burns at a lower temperature than glass melts, if you attempt to seal the dirty side first it will burn, expand, and occasionally blow your sample out of the clean end. You should therefore always seal the clean end of the capillary first.
The procedure is no different from sealing with water in the tube. However, with the dirty end you will find it significantly more difficult to seal. To be successful you need to hold the capillary in the flame marginally longer, however, it is easy to wait too long and end up with glass bubbles. If you continue to have problems, sealing wax can be used as a second protective measure once the capillaries have been flame sealed (candle wax is not a suitable substitute for sealing wax). Heat the wax in the torch flame until it is molten. Quickly dip and roll the capillary end in the molten wax and hold the capillary with the wax covered end pointing downwards so that the wax covers the end of the capillary – it should look like a match head when it hardens. If you do not hold the wax end downwards as it hardens the wax will run away from the end of the capillary and it will not be effective. You need to use enough wax to completely cover the end of the capillary but not so much that there is a huge blob of wax present otherwise the capillaries will not fit in the distillation tubes when the samples are sent for analysis.
The picture below shows a well sealed capillary (top) with an air gap at each end of the blood sample. The blood has travelled to one end in the bottom capillary, which often indicates that there is a hole in the end (even if you can't see it). It is likely that the bottom capillary will dry out over time.
The picture below shows a capillary that has dried out. It is impossible to get results from samples that have dried out.
A close-up of the end of a well sealed capillary - the end should be fairly rounded, not pointed enough that it feels sharp like a thorn. There should be no long glass threads or glass bubbles.
The picture below shows a well sealed capillary with a rounded end (bottom) compared to one with a fine thread-like point (top), which may break off leaving a hole in the end, and a capillary with a bubble in the glass (middle), which may be fragile and liable to break.
The picture below shows wax that has been properly applied to the end of a capillary (bottom) alongside one where the capillary has been turned before the wax has hardened (top). The wax on the top capillary has moved away from the end (the point of the capillary can be seen) and will not be effective at preventing poorly sealed capillaries from drying out. It is possible to see that this is the case here - the blood in the top capillary has moved towards this end and there is no air gap at the end as there is in the bottom capillary.
The capillaries need to fit into our distillation tubes (below left) - too much wax on the ends prevents this from happening (below right). Only one end of the capillary is cut, so if there is too much wax on both ends of the capillary we have to remove some wax by heating it - this risks heating the blood sample.
